L-lysine is an essential amino acid that is not synthesized in animals. Many wild type and mutant bacterial strains have been found to produce L-lysine. Being widely used as a feed additive, medicament, chemical agent and food ingredient, L-lysine has been produced by large-scale fermentation using mainly a Coryneform bacterium or an Escherichia bacterium.
In most bacteria, L-lysine is naturally synthesized from aspartate in a nine-step enzymatic pathway, including two steps shared by the biosynthesis pathways of methionine and threonine (Anastassiadis, S., Recent patents on Biotechnology 2007, 1(1):11-24; Chen, N. et al., J. Biol. Chem. 1993, 268(13):9448-66). The regulatory mechanism of lysine biosynthesis is complex and varies widely in different bacterial species (Chen, N. et al., J. Biol. Chem. 1993, 268(13):9448-66). For example, dihydrodipicolinate synthase (“DDPS”), an enzyme that catalyzes the first step into the lysine biosynthesis branch, suffers feedback inhibition by L-lysine in Gram-negative bacteria (e.g., E. coli, Bacillus sphaericus and Methanobacterium thermoautotrophicum), but not in Gram-positive bacteria (e.g., Bacillus licheniformis, Bacillus megaterium, Bacillus subtilis, Corynebacterium glutamicum, Bacillus cereus, and Bacillus lactofermentum) (Dobson, R. et al., Acta Cryst. 2005, D61:1116-24). Further, the regulation of the lysine biosynthesis pathway in Bacillus subtilis (“B. subtilis”) is unique because it involves a dual control by lysine and one of its precursors, diaminopimelate (Chen, N. et al., J. Biol. Chem. 1993, 268(13):9448-66).
Consistent with the diverse sensitivity to feedback inhibition of DDPS by L-lysine, limited homology in the DDPS protein sequence and in its corresponding gene, dapA, is observed among bacterial strains from different genera. DDPS in B. subtilis has an amino acid sequence about 43% and 40% identical to those in E. coli and Corynebacterium Glutamicum (“C. Glutamicum”), respectively (Chen, N. et al., J. Biol. Chem. 1993, 268(13):9448-66). Even in the same bacterial genus, different bacterial strains exhibit only modest homology. For example, the dapA gene in Bacillus methanolicus (“B. methanolicus”) is about 65% identical in nucleotide or amino acid sequence to a previously known dapA gene in B. subtilis (U.S. Pat. No. 6,878,533).
One way to improve L-lysine production by an Escherichia bacterium is to overcome the feedback inhibition of DDPS by L-lysine. Mutations have been made in the wild type dapA gene of an Escherichia bacterium to desensitize DDPS to L-lysine (U.S. Pat. No. 6,040,160). Attempts have also been made to introduce a wild type dapA gene of a non-Escherichia bacterium, in which the corresponding DDPS does not suffer feedback inhibition by L-lysine, into an Escherichia bacterium, but have failed to produce consistent and satisfactory results.
A Korean group reported that an introduction of a wild type dapA gene from a lysine overproducing C. glutamicum strain into a lysine producing mutant E. coli strain (TF1) led to a parallel increase of a lysine-sensitive DDPS activity and lysine production (Oh, J. et al., Biotech. Ltrs. 1991, 13(10):727-32; Korean Pat. Pub. No. 10-1992-0008382). However, expression of the same wild type dapA gene in two other E. coli strains (TF13 and TF23) failed to result in a high yield of lysine production. The fact that the regulatory mechanism involved in lysine biosynthesis is more complex in E. coli than in Coryneform bacteria was cited for the inconsistent results.
Expression of a foreign dapA gene is challenging because the corresponding foreign DDPS protein is likely subject to decomposition by protease and formation of an insoluble inclusion body in an Escherichia bacterium (U.S. Pat. No. 6,040,160). In addition, a DDPS of C. glutamicum (Oh, J. et al., Biotech. Ltrs., 1991, 13(10):727-32; Korean Pat. Pub. No. 10-1992-0008382) or B. methanolicus (U.S. Pat. No. 6,878,533) is not expected to exhibit its advantageous activity, i.e., a lysine-insensitive DDPS activity that leads to a high yield of lysine production, in E. coli partly because the optimal cultivation temperature for C. Glutamicum or B. methanolicus deviates from that for E. coli by about ten or more degrees.
An extremely complicated regulation of lysine synthesis was observed in E. coli cells, in which genes involved in lysine biosynthesis in B. subtilis were expressed (Shevchenko, T. N. et al., Tsitol Genet. 1984, 18(1):58-60). In particular, the expression of these foreign genes, including a foreign dapA gene, in E. coli cells failed to increase lysine production to a high and satisfactory level. It was suggested that a considerable increase in lysine biosynthesis be achieved by using an E. coli or B. subtilis strain having mutations in its natural genes involved in lysine biosynthesis to desensitize feedback inhibition by lysine and diaminopimelate.
At present, there has not been any effective method for producing L-lysine using an Escherichia bacterium comprising a wild type or variant B. subtilis dapA gene. As the demand of L-lysine, especially for animal feed, continuously increases along with the global population expansion, there is a need to develop novel and effective methods for improving L-lysine production using an Escherichia bacterial strain.